This is a new research proposal aiming at studying purine and pyrimidine metabolism in the anaerobic flagellate protozoan parasites Trichomonas and Giardia both causing serious human health problems around the world. The reason behind this interest is based on preliminary data on enzyme profiles indicating a lack of de novo biosynthesis of both purines and pyrimidines in these organisms. Only a few unique enzyme activities of purine and pyrimidine salvage were detected in the parasites, suggesting that selective inhibition of some of these enzymes may provide effective chemotherapeutic control of the parasitic infections. What we are planning to do is to cultivate in vitro T. vaginalis, G. lamblia and a closely related model cattle parasite Tritrichomanas foetus. The log-phase cells will be pulse-labeled with radioactive purines, pyrimidines and their nucleosides. Labels incorporated into the nucleotide pool will be extracted and analyzed in high pressure liquid chromatography. Data will be compared with those from enzyme profile studies, and used to construct a complete map of purine and pyrimidine metabolisms in the three parasites. Pivotal enzymes in purine and pyrimidine salvage will be identified, purified by affinity column chromatography and characterized. Inhibitors of these enzymes, such as the UPRTase inhibitor emimycin, will be examined on their detailed mechanisms of inhibition, their probable effects on pulse-labeling of nucleotide pools in the parasites and their probable inhibition of in vitro growth of the parasites. Mutants resistant to the inhibitors will be isolated by mutagenising the cells and selecting cell clones on agarose plates to see (1) if it represents lethal mutation; or (2) if there is alteration in the target enzyme and the metabolic pathway as the basis of resistance. In all, our short-term aim is to understand the purine and pyrimidine metabolisms in Trichomanas and Giardia. Our long-term interest is to take advantage of the defects in the parasite purine/pyrimidine metabolism for a rational approach to chemotherapeutic control of these parasites.